Imaging mass spectrometry of histologic thin tissue sections, or other two-dimensional samples, with ionization of the molecules of interest by matrix-assisted laser desorption (MALDI) has recently experienced an upsurge. The usual procedure is to measure the distributions of certain proteins, which, either alone or in combination with other proteins, can serve as biomarkers to characterize the stress or disease status of individual parts of the thin tissue section. A method of this type is described in U.S. Patent Publication 2006/0063145.
The prior art method requires the thin tissue section be coated with a layer of small matrix crystals in a special way, so that there is a high degree of ionization of the proteins and other substances of interest. A coating method of this type is described in German patent publication DE 10 2006 019 530.2.
Using specially shaped laser beam profiles, such as those described in U.S. Pat. No. 7,235,781, for example, it is possible to achieve spatial resolutions of approximately 50 micrometers when measuring molecular distributions in thin sections. This is sufficient for most applications. However, to obtain a good measurement with high sensitivity and a sufficiently high accuracy for the concentration measurement, it is not sufficient to scan an individual mass spectrum; between 50 and 500 individual spectra have to be added together to form a sum spectrum. In order to fully utilize the spatial resolution by using a measurement grid spacing of 50 micrometers, 40,000 sum spectra per square centimeter of thin section must be scanned, and these sum spectra must be assembled from several million individual spectra. It follows that the scanning time for an individual sum spectrum is crucial for the practicability of the method. Obviously, lower spatial resolutions can also be chosen. For body cross-sections of mice or rats, for example, very good distributions of the analyte substances over the individual organs and spaces between organs can be measured with grid spacings of between 200 and 500 micrometers. This only requires scanning of 2,500 and 400 sum spectra per square centimeter respectively. Nevertheless, these sum spectra can still include between a hundred thousand and a million individual spectra. Even in this case it is desirable to have a high scanning frequency with preferably more than 1,000 individual spectra per second.
Quite often interest is not, however, focused only on proteins and other macromolecules, but increasingly on the distribution of smaller molecules such as drugs or their metabolites in the tissue areas of the thin sections. The accumulation of drugs and their metabolites in certain organs or in certain kinds of tissue, for example tendons, connective tissues, nerves, muscle fibers and arterial or venous blood vessels, is of great interest when studying the effectiveness of these drugs. These small molecules generally have molecular weights roughly in the range 150-500 Daltons and thus lie within a mass range, which, in MALDI time-of-flight mass spectrometry, suffers such a high degree of interference from ions of complexes of the matrix substance and their fragments that good detection sensitivity cannot be achieved. Every single mass on the mass scale is already occupied by several different species of complex ions, thus creating a strong chemical background noise, which interferes with, or even prevents, any sensitive measurement of small molecules.
One solution to this dilemma is to measure a selected daughter ion from the fragmentation of the molecular ions of this small molecule, rather than the molecular ions themselves. These methods, which serve to improve the signal-to-noise ratio and also to increase the specificity of the determination, are already familiar in other fields of mass spectrometry under the abbreviation SRM (selective reaction monitoring). Modern tandem time-of-flight mass spectrometers such as those described in U.S. Pat. No. 6,300,627 are available to measure the daughter ions. These tandem time-of-flight mass spectrometers comprise two time-of-flight mass spectrometers in sequence, and are generically referred to by the abbreviation “TOF-TOF”. The first time-of-flight mass spectrometer is used to select the parent ions; the second one measures the daughter ions resulting from the fragmentation of the parent ions. The fragmentation can be brought about by using a slightly stronger laser irradiation during the MALDI process, thereby creating metastable ions, which decompose in flight, or the fragmentation may be generated by collisions in gas-filled collision chambers.
Instead of using TOF-TOF-instruments, the daughter ions can also be measured with an instrument which uses the MALDI process for ion generation, then generates the daughter ions from the analyte ions by collision processes and detects them in a time-of-flight mass spectrometer with orthogonal ion injection. These instruments are, however, similarly expensive to tandem time-of-flight mass spectrometers.
Tandem time-of-flight mass spectrometers (TOF-TOF) have almost completely superseded the earlier customary method of measuring daughter ions in simple time-of-flight mass spectrometers with reflectors, which became known as “PSD” (post source decay), because they offer a significantly improved mass resolution, better substance utilization and far easier operation. Although it was possible to obtain the PSD spectra using relatively low-cost reflector time-of-flight mass spectrometers, they had to be compiled from 12 to 15 partial spectra, each of which had to be obtained individually by a new ionization of sample, using methods which were complicated to control. But modern tandem time-of-flight mass spectrometers also have disadvantages. They are relatively expensive and, for electronic reasons, cannot yet offer a high scanning frequency for the measurement of daughter ions.